Necessity of detergent for efficient puromycin-mediated release of nascent peptides from rat liver ribosomes.
Puromycin-mediated in vitro release of nascent peptides from rat liver ribosomes was significantly stimulated by the presence of low concentrations of a detergent, and the stimulation was much more marked with bound ribosomes than with free ribosomes. The release of nascent peptides from ribosomes could be carried out in two steps, first with puromycin in the absence of a detergent and then with a detergent, to give two separate nascent peptide fractions S1 and S2, respectively. Although S1 and S2 fractions were not significantly different in hydrophobicity and in the size of the puromycin-conjugated peptides when examined by alkyl-Sepharose column chromatography and SDS-polyacrylamide gel electrophoresis, the fractionation of the released peptides by immunoprecipitation showed significant difference in the distribution of the nascent peptides of two specific proteins, serum albumin and NADPH-cytochrome c reductase, between these two fractions. The nascent peptides of serum albumin were found mainly in fraction S1 obtained from bound ribosomes. On the other hand, a larger portion of the nascent peptides of NADPH-cytochrome c reductase was detected in fraction S2 from free ribosomes than in other fractions. The presence of a detergent is indispensable for efficient in vitro release of nascent peptides from ribosomes by puromycin and this finding may be important in studying the synthesis of specific proteins in mammalian cells.
D000818:Animals; D003902:Detergents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D008297:Male; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis; D010455:Peptides / Q000302:isolation & purification*; D011691:Puromycin / Q000494:pharmacology*; D051381:Rats; D012269:Ribosomal Proteins / Q000302:isolation & purification*; D012709:Serum Albumin / Q000032:analysis; D013501:Surface-Active Agents / Q000494:pharmacology*
D003902:Detergents; D010455:Peptides; D012269:Ribosomal Proteins; D012709:Serum Albumin; D013501:Surface-Active Agents; D011691:Puromycin; D009251:NADPH-Ferrihemoprotein Reductase